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Methods Mol Biol. 2015;1208:63-71. doi: 10.1007/978-1-4939-1441-8_5.

Nitric oxide availability as a marker of oxidative stress.

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Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, The University of Texas Graduate School of Biomedical Sciences at Houston, The University of Texas Health Science Center at Houston, Houston, TX, USA.


Nitric oxide (NO) is widely considered one of the most important molecules produced in the human body, acting as a necessary regulator in a vast array of vital physiological functions, namely, blood pressure, immune response, and neural communication. Healthy endothelium is defined by the ability to produce adequate levels of NO. Reactive oxygen species (ROS) play a major role in NO-based cell signaling. ROS can affect NO availability both from production to post-production scavenging and lead to a myriad of vascular disorders due to compromised NO functionality. In 2004, it was identified in animal models that oxidative stress plays a significant role in the development of hypertension, in part by inactivation of NO (Ghosh et al., Br J Pharmacol 141(4):562-573, 2004). It was thus concluded that NO bioavailability was reduced in the presence of ROS. We speculated that the accurate detection of NO and quantification in biological matrices is critical as a marker of oxidative stress (Bryan et al., Proc Natl Acad Sci USA 101(12):4308-4313, 2004). The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively, and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone-based chemiluminescence with chemical derivatization to determine molecular source of NO as well as ex vivo with organ bath myography. This approach ties fundamental biochemistry to functional response.

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